| Glucosinolate is an important secondary metabolite in cruciferous plants,and its hydrolysate plays an important role in anti-nutrition of the human loeing.Glucosinolates are mainly synthesized in young and tender parts of plants,and transferred to siliques,seeds and other parts for storage during the reproductive growth period.The glucosinolate content is different in different parts and different periods.In recent years,with the development of technology,people have a more in-depth understanding of the biosynthesis and degradation pathways of glucosinolates,but the mechanism of glucosinolate transport is still unclear.Therefore,in-depth study of the regulation mechanism of glucosinolates in pak choi is of great significance to the breeding of pak choi and even cruciferous vegetables.In this experiment,HPLC was used to determine the glucosinolate components and their contents in different parts of pak choi,and the total glucosinolate content of stem apex at different developmental stages was determined by ELISA.In order to analyze the molecular mechanism of the change of glucosinolate content,using the brassica rapa subsp.pekin genome as a reference,expression profile of the stem apex at vegetative growth period(10d after transplanting),the critical period of flower bud differentiation,namely flower bud differentiation stage 0 and flower bud,defferentiation stage 1,3 and 5 were analyzed by RNA-Seq technology,so as to clarify the reason for the change in glucosinolate content.The results in details are as follows:1)The content of glucosinolates in different parts of pak choi was determined by HPLC.The results showed that the highest total glucosinolate content of the 9 parts was the vegetative roots,followed by the reproductive roots and siliques,and then the tender leaves.The total glucosinolate content in Silique higher than stalk and flower bud.Each of the 9 parts contains 8 glucosinolate components,among them,aromatic glucosinolates were not detected in these 9 parts.Eli SA was used to determine the total glucosinolate content of the shoot apex at different developmental stages,and it was found that the total glucosinolate content in the vegetative growth phase was higher than the critical period of flower bud differentiation and the stage of flower bud differentiation.With the flower bud differentiated,the total glucosinolate content becomes higher and higher.2)Transcriptome sequencing was performed on 15 samples in 5 periods,and a total of111.44 Gb Clean Data was obtained.The GC content in each sample was above 47%,and the base Q30 was more than 94%.The high-quality total reads obtained from each library were compared with the brassica rapa subsp.pekin genome database,and the sample comparison rate was between 85.74%-87.61%,the efficency is higher.3)Pairwise comparison analysis of transcriptome data showed that a total of 3058 DEGs were obtained from 15 samples in 5 periods.The results of functional enrichment analysis of the DEGs GO showed that the biological processes of amino acid synthesis,secondary metabolite synthesis and photosynthesis were significantly enriched in different developmental stages of the stem apex.KEGG enrichment pathway analysis of DEGs found that tryptophan metabolism,phenylpropane biosynthesis,glucosinolate biosynthesis and other pathways in different developmental stages of the stem tip were annotated.4)Using bioinformatics methods,the NPF gene family of pak choi was analyzed.The results showed that there are 72 members in the NPF gene family of pak choi,which are divided into 8 sub-families.The number of amino acids is between 497-1117 aa,most of which are stable basic proteins.The most secondary structures are α-helices,and the subcellular locations are all located on the plasma membrane.The NPF gene of pak choi is unevenly located on 10 chromosomes,most of which contain 4 CDS structures. |
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